Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds. The general structure of an antibody of class IgG (i.e. an immunoglobulin (Ig) of class gamma (G) is shown schematically in FIG. 1A.
Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain.
Antigen binds to antibodies via an antigen binding site in the variable domains of each pair of light and heavy chains. Other molecules, known as effector molecules, bind to other sites in the remainder of the molecule, i.e. other than the antigen binding sites, and this portion of antibody will be referred to as “the constant portion” of an antibody, such sites being located particularly in the Fc region constituted by the portions of the heavy chains extending beyond the ends of the light chains.
The constant portion of antibodies specifically interact with receptors on “effector” cells. For example, the Fc region mediates effector functions that have been divided into two categories. In the first are the functions that occur independently of antigen binding; these functions due to the major histocompatibility complex class I-related receptor FcRn confer IgGs persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis (see Ghetie V and Ward S). In the second are the functions that operate after an antibody binds an antigen; these functions involve the participation of the complement cascade or Fc receptor (FcR) bearing cells.
FcRs are defined by their specificity for immunoglobulin isotypes. For example Fc receptors for IgG antibodies are referred to as FcγR. FcRs are specialized cell surface receptors on hematopoietic cells that mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody.
The FcγRs play a critical role in either abrogating or enhancing immune recruitment. Currently, three classes of FcγRs are distinguished on cells of the immune system: the high-affinity receptor Fc RI (CD64), capable of binding monomeric IgG; and the low-affinity receptors FcγRII (CD32), and FcγRIII (CD16), which interact preferentially with complexed IgG. Furthermore, the FcγRII and FcγRIII classes comprise both “A” and “B” forms (Gessner-J E et al. Ann Heamatol, 1998, The IgG Fc receptor family, 76: 231-248).
FcγRII proteins are 40 KDa integral membrane glycoproteins which bind only the complexed IgG due to a low affinity for monomeric Ig (106 M−1). This receptor is the most widely expressed FcγR, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. Fc γRII has only two immunoglobulin-like regions in its immunoglobulin binding chain and hence a much lower affinity for IgG than FcγRI. There are three human FcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG in aggregates or immune complexes. The gene for the FcγRIIa receptor contains either G or A in codon 131, resulting in either arginine (CGT) or histidine (CAT), respectively, in the second extracellular domain. This change alters the ability of the receptor to bind IgG. Cells with FcγRIIA His-131, the A/A genotype, bind human IgG2 with considerably higher affinity than those with Arg at position 131; conversely, cells with Arg-131, the G/G genotype, bind murine IgG1 with considerably higher affinity than those with His at position 131 (Salmon et al., 1992, J. Clin. Invest. 89:1274-1281). Originally, studies using monocytes interaction with an anti-CD3 antibody of the murine IgG1 subclass as a trigger for T-cell proliferation classified individuals phenotypically as low responders of high responders (Tax et al., 1983, Nature: 304: 445-447). It is now known that high responder cells in this assay have the G/G or A/G genotype while low-responder cells have the A/A genotype. The FcγRIIa 131 R/R genotype is a risk factor for susceptibility to some infectious and autoimmune diseases (Van der Pol W. L. and Van de Winkel J. G. J, 1998, Immunogenetics 48:222-232).
Distinct differences within the cytoplasmic domains of FcγRII-A and FcγRII-B create two functionally heterogeneous responses to receptor ligation. The fundamental difference is that the A isoform initiates intracellular signaling leading to cell activation such as phagocytosis and respiratory burst, whereas the β isoform initiates inhibitory signal, e.g., inhibiting B-cell activation.
Monoclonal antibodies (mAbs) have now been used to treat disease in human patients. Although some mAbs may function effectively without utilizing antibody effector functions (e.g. neutralizing antibodies), in many cases it may be desirable to engineer the Fc portion of the antibody to recruit the immune system to elicit an immune response. There exists a need in the art to produce antibodies that include a variant Fc region having increased potency while retaining the ability to bind to a given target. Accordingly, there exists a need to produce altered IgG antibodies that elicit a modified Fc receptor activity while retaining binding to an antigen as compared to an unaltered antibody.